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Study of Clusterin : an Extracellular Chaperone Protein Stephen Poon University of Wollongong

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Study of Clusterin : an Extracellular Chaperone Protein Stephen Poon University of Wollongong University of Wollongong Research Online University of Wollongong Thesis Collection University of Wollongong Thesis Collections 2002 Study of Clusterin : an extracellular chaperone protein Stephen Poon University of Wollongong Recommended Citation Poon, Stephen, Study of Clusterin : an extracellular chaperone protein, Doctor of Philosophy thesis, Department of Biological Sciences, University of Wollongong, 2002. http://ro.uow.edu.au/theses/1056 Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: [email protected] STUDY OF CLUSTERIN: AN EXTRACELLULAR CHAPERONE PROTEIN. A thesis submitted in fulfilment of the requirements for the award of the degree of Doctor of Philosophy From THE UNIVERSITY OF WOLLONGONG By Stephen Poon (B.Sc. Hons) Supervisors - Mark R. Wilson* and John A. Carver^ Department of Biological Sciences t Department of Chemistry 2002 DECLARATION OF AUTHENTICITY This thesis is submitted in accordance with the regulations of the University of Wollongong in fulfillment of the requirements for the degree of Doctor of Philosophy (PhD). It does not incorporate any material previously published or written by any person except where due reference is made in the text. The experimental work described in this thesis is original work and has not been previously submitted for a degree or diploma at any other university. STEPHEN POON 11 \ ACKNOWLEDGEMENTS I wish to acknowledge, with the utmost appreciation and thanks, the following who have shown great professionalism, patience and guidance over the course of my post-graduate degree: Associate Professor Mark R. Wilson, Department of Biological Sciences; and Associate Professor John A. Carver, Department of Chemistry; both of University of Wollongong, Wollongong, NSW, Australia. Without them, this thesis would not have been accomplished. I would also like to express my thanks and gratitude to Professor Martin Tenniswood for putting up with me during an unforgettable term at the University of Notre Dame, Indiana, USA. I would also like to extend my thanks to my parents for their love and continual support throughout my degree. They encouraged me to aim high, to jump over every hurdle, break through all barriers, and to strive to be the best! Thanks for such invaluable advice. A special thanks also to my mentor, Dr. David Humphreys (aka, "clusterin man"), whose guidance during my Honours and parts of Post-Graduate degrees have been momentous; and to Robyn A. Lindner for her forthcoming support. Finally, I would like to thank all my fellow students (past and present) for their friendship and understanding. In particularly, I would like to acknowledge Alison Smail, Rachel Jones, Christine Gillen, and Teresa Treweek, for their jovial behaviour; without you guys, my time at Wollongong would have been very boring. I wish you all the best for the future and thanks for the memories. To Elise Stewart and Justin Yulbury, keep pushing the frontiers of clusterin research forward. I expect to see you publishing at least one paper a year! 111 ^x Finally, a special mention of thanks to those with whom I have had the pleasure to be acquainted whilst at Notre Dame. To Kerry Gilmore, for letting me stay with you; to Louise Flanagan, for being such a great friend, and to the following for accepting me into their 'Irish/Canadian/American community'; Lorna, Mark, Edmund, Gerry, Kathryn, Kenneth, Soma, Sharon, Glendon, Judy, Sarra, Emma, Kathleen, and Uwe. Love you all! STEPHEN POON IV \ ABSTRACT Clusterin is a widely distributed and highly conserved secreted mammalian glycoprotein whose elevated expression has been detected in a number of disease states (e.g. Scrapie, Alzheimer's and Creutzfeldt-Jakob diseases) that are associated with abnormally high levels of misfolded and/or precipitated proteins. Clusterin, present at concentrations ranging from 35-105 ug/ml in human serum, is known to interact with a variety of proteins and lipids in vitro. These numerous binding interactions have led to a number of biological functions being proposed for clusterin; these include roles in reproduction, lipid transport, endocrine secretion, apoptosis, and complement regulation. In a recent study, it was reported that clusterin has molecular chaperone activity in vitro. Molecular chaperones, as defined by John Ellis, are proteins that function in vivo to specifically interact with and stabilise unfolded or partially unfolded proteins, thereby preventing them from potentially aggregating and precipitating, e.g. during protein folding. In this study, clusterin was shown to prevent the precipitation of heat-stressed ovotransferrin and y-crystallin, as well as DTT-reduced ovotransferrin and lysozyme. Analysis by size exclusion chromatography of samples in which clusterin was co-incubated with ovotransferrin or lysozyme undergoing stress-induced denaturation revealed the presence of HMW species in the void volume that was eluted from the column. Subsequent analysis by SDS-PAGE of these HMW species, confirmed the presence of both clusterin and the stressed target protein. Results presented in this report also demonstrate that clusterin protects proteins in (i) diluted human serum from heat-induced precipitation and (ii) undiluted human serum from DTT-mediated precipitation. Other results presented in this thesis indicate that clusterin does not have the ability to hydrolyse ATP and hence, performs V its chaperone function in an ATP-independent manner. In addition, clusterin was unable to independently facilitate the reactivation of heat-inactivated ADH and catalase after removal of stress. However, in the presence of a chaperone with refolding capability (i.e. Hsc70) and ATP, clusterin-stabilised ADH and catalase were partially refolded. Taken together, these results raise the possibility that clusterin may inhibit precipitation of human serum proteins in vivo and create a reservoir of inactive protein structures from which folding-competent proteins can be subsequently reactivated by other refolding chaperones. Clusterin was shown to inhibit the slow precipitation of y-crystallin and lysozyme, but was unable to prevent these same target proteins from rapid precipitation. Real-time H NMR spectroscopic analysis of the interaction between clusterin and a-lactalbumin reveal that clusterin did not alter the rate of a-lactalbumin reduction but did stabilise the less ordered intermediately folded form of the protein. These results suggest that (i) kinetic factors are important in the chaperone action of clusterin and (ii) clusterin binds specifically to slowly aggregating proteins on the irreversible off-folding pathway. This thesis also presents results to show that elevated temperature (up to 50 °C) does not induce significant changes in the oligomerisation state of clusterin nor does it substantially alter the ability of clusterin to interact with heat-stressed or chemically reduced target proteins. In contrast, incubation at mildly acidic conditions resulted in the dissociation of clusterin oligomers, which led to an increased exposure of hydrophobic regions on clusterin to solution and a concomitant enhancement of its chaperone action. A phenomenon known as acidosis occurs at sites of tissue damage or inflammation where the local pH can drop below 6. Acidosis has been reported to occur at sites of inflammation as well as in many of the diseases to which clusterin has been associated. Taken together, these results vi suggest that under these conditions, the dissociation and enhanced chaperone actions of clusterin could help to inhibit the aggregation and deposition of inflammatory and/or toxic insoluble protein deposits which would otherwise exacerbate pathology. At present, the structural regions responsible for the ligand binding and chaperone action of clusterin have not been identified. Sequence analysis of clusterin has revealed several regions that could be functionally important, including three regions of amphipathic a-helices and two coiled-coil domains. In addition, studies have shown that, despite having variable truncations at the C-terminus of the a-chain and the N-terminus of the (3-chain, clusterin expressed by yeast Pichia pastoris has similar chaperone activity to human clusterin in vitro, indicating that the sites responsible for the chaperone action of clusterin are more likely to be located more towards the N-terminal region of the a-chain and the C- terminal region of the [3-chain. In order to identify the functional sites of clusterin and to test the above statements, five proline-substitution and five truncation clusterin mutants, as well as wild-type clusterin, were developed. These mutants, including wild type clusterin, were expressed in transiently transfected Spodoptera frugiperda (Sf9) insect cells. Sf9 cells were chosen as the host for the expression of wild type and mutant clusterin due to their reported ability to express high levels of recombinant proteins and perform post-translational modifications in a manner that is similar to mammalian cells. However, Western blot analysis showed that the expressed proteins were not expressed at high levels and had molecular masses that were approximately 15 kDa smaller than their expected sizes. Since the Sf9 transfectants were not cloned, the Sf9 cell cultures may have contained a large number of non-transformants/non-secretors that may have outnumbered secreting transformants and hence, explain why the yield was so low. The size irregularity can
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